US8999937B2 - Glucocorticoid induced leucine zipper mimetics as therapeutic agents in multiple sclerosis - Google Patents

Glucocorticoid induced leucine zipper mimetics as therapeutic agents in multiple sclerosis Download PDF

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US8999937B2
US8999937B2 US14/001,716 US201214001716A US8999937B2 US 8999937 B2 US8999937 B2 US 8999937B2 US 201214001716 A US201214001716 A US 201214001716A US 8999937 B2 US8999937 B2 US 8999937B2
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gilz
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pharmaceutical composition
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Mythily Srinivasan
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Indiana University Research and Technology Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • the invention pertains generally to compositions that mimic the activity of glucocorticoid induced leucine zipper (GILZ) on the immune system. More particularly, the invention pertains to the use of GILZ mimetics for treatment of multiple sclerosis.
  • GILZ glucocorticoid induced leucine zipper
  • Glucocorticoids act by interacting with the glucocorticoid receptor (GR) in the cytoplasm, which then translocates to the nucleus and binds the GR responsive elements (GRE) in the promoter regions of target genes.
  • GILZ Glucocorticoid induced leucine zipper
  • GILZ is a glucocorticoid-inducible gene with six GRE elements.
  • Overexpression of GILZ suppresses activated T cells by inhibiting transactivation of nuclear factor kappa B (NF- ⁇ B), the master regulator of inflammatory responses.
  • NF- ⁇ B nuclear factor kappa B
  • the anti-inflammatory activity of synthetic glucocorticoids mediated by preventing NF- ⁇ B transactivation has been attributed to the induced upregulation of GILZ (SEQ ID NO: 1).
  • MS Multiple sclerosis
  • NEMO NF- ⁇ B essential modulator
  • NBD NEMO binding unit
  • MS is widely recognized as a complex disease driven by dysregulated immunity accompanied by relapsing and remitting clinical manifestations. Since the antigenic trigger of MS is not definitively identified, most disease modifying agents are designed to modulate the inflammatory response in the periphery and in the brain. Therapies under development include strategies that deplete lymphocytes and those that mediate immunomodulation. Since depletion could potentially cause global immunosuppression, strategies of immunomodulation are preferable.
  • GILZ originally identified as a glucocorticoid-inducible gene, has been shown to interact with the p65 subunit of NF- ⁇ B in activated T cells. Since activated p65 is present only in stimulated cells, the GILZ mimetics may mediate selective inhibition of activated cells without causing widespread immunosuppression. Preliminary results suggest that treatment with exogenous GILZ (SEQ ID NO: 1) suppresses antigen activated T cell responses in vitro. Low molecular weight GILZ mimetics that bind the p65 protein with optimal kinetics are characterized. Advantages of peptides such as GILZ mimetics as therapeutics include non-immunogenicity with potential for long-term use, greater permeability to cross tissue barriers, and cost-effectiveness.
  • glucocorticoids One of the actions of glucocorticoids is to modulate the transcription of multiple genes involved in immune response.
  • the anti-inflammatory effect is largely attributed to the inhibition of nuclear factor kappa B (NF- ⁇ B), a regulator of inflammatory responses.
  • GILZ having an amino acid sequence MNTEMYQTPMEVAVYQLHNFSISFFSSLLGGDVVSVKLDNSASGASVVAIDNKIEQAM DLVKNHLMYAVREEVEILKEQIRELVEKNSQLERENTLLKTLASPEQLEKFQSCLSPEEP AP ESPQVPEAPGGSAV (SEQ ID NO: 1), is a glucocorticoid induced gene that binds the p65 subunit of NF- ⁇ B to inhibit its nuclear translocation.
  • NF- ⁇ B is a heterodimer of p50 and p65 that remains as an inactive complex with inhibitory proteins such as I ⁇ B in the cytoplasm of resting T cells. Following T cell activation, p65 is released from the inhibitory complex, translocates to the nucleus and mediates transactivation of inflammatory genes.
  • Gene profiling studies revealed the presence of elevated p65 in the peripheral blood monocytes and in the pathologic lesions in MS. It is believed herein that therapeutic agents that sequester activated p65 within the cytoplasm will suppress transactivation of inflammatory cytokines and ameliorate disease in MS.
  • proline rich carboxy terminus of GILZ (SEQ ID NO: 1) interacts physically with the p65 subunit of NF- ⁇ B.
  • Proline rich regions PRR
  • PRRs are often localized in the solvent exposed regions of proteins involved in transient interactions such as signaling or cytoskeletal rearrangements.
  • PRRs provide target sites for developing inhibitors of transient protein-protein interactions.
  • Rationally designed peptide mimetics of the proline rich p65 binding interface of GILZ (SEQ ID NO: 1) may sequester p65 within the cytoplasm in activated T cells and suppress inflammation related to MS. Without being bound by any particular theory, such peptide mimetics may act as a glucocorticoid mimetic and/or as an NF- ⁇ B inhibitor.
  • GILZ-COOH The carboxy terminus of GILZ (GILZ-COOH) consists of 35 residues (residues 100-134 of GILZ); LASPEQLEKFQSCLSPEEPAPESPQVPEAPGGSAV (SEQ ID NO: 2).
  • PEEP SEQ ID NO: 3
  • PESP SEQ ID NO: 4
  • PEAP SEQ ID NO: 5
  • the role of PRRs is to bring proteins together so as to make subsequent interactions more probable. This is particularly true for interactions between functionally important proline and conserved hydrophobic residues in the interface of its binding partner.
  • the transactivation domain of p65 that potentially interacts with the GILZ protein presents two highly conserved phenylalanine residues; F534 and F542 which together with the conserved acidic residues at Asp 531 and Asp 533 and phosphorylation sites at Ser 529 and Ser 536 constitute the critical residues for p65 transactivation.
  • GILZ mimetics Data from these observations are integrated with that from residue interface propensity to introduce rational amino acid substitutions and/or truncations in the GILZ-WT so as to design GILZ mimetics with optimal p65 binding efficacy. Additional parameters considered to increase the drug like properties of GILZ mimetics include solvent mediated contact potential, accessible surface volume/residue, log interface residue propensity, weighted hydrophobicity and solvation potential.
  • the proof of therapeutic manipulation of specific intermolecular interactions is derived from studies using fusion proteins and/or monoclonal antibodies of the interacting molecules such as Abatacept (CTLA4 IgG1Fc), Alefacept (LFA-3-IgG1Fc), Denileukin diftitox (recombinant IL-2) and Etanercept (TNFR-IgG1Fc) or adalimumab/Certolizumab pegol/infliximab (anti-TNF- ⁇ mAb), Natalizumab (anti- ⁇ 4 integrin,) and Efalizumab (anti-CD11a mAb) respectively.
  • CTL4 IgG1Fc Abatacept
  • Alefacept LFA-3-IgG1Fc
  • Denileukin diftitox recombinant IL-2
  • Etanercept TNFR-IgG1Fc
  • GILZ:p65 interaction occurs in the cytoplasm necessitating intracellular delivery of potential modulating agent(s).
  • low molecular weight peptides that are permeable and better amenable for intracellular delivery than large proteins represent attractive alternatives.
  • GILZ mimetics will suppress pro-inflammatory responses by sequestering activated p65 and facilitate skewing towards anti-inflammatory responses.
  • An advantage of lower molecular weight peptides as therapeutic agents include increased permeability for intracellular delivery compared to larger proteins.
  • a pharmaceutical composition comprising a polypeptide from about 6 to about 35 amino acid residues, the polypeptide comprising 1 to 3 tetrapeptides having the sequence of PXXP, wherein
  • cell penetrating peptide is selected from the group consisting of Penetratin, Pep-1, Pep-2, VP22, pVEC, pISL, hCT derived peptide, LL-37, Mouse PrP, Transportan, TP10, Arg11, MAP, MPG, KALA, ppTG1, and ppTG20.
  • cell penetrating peptide is a bipartite peptide (K16ApoE) consisting of a polylysine segment linked with the apolipoprotein-E peptide.
  • cytokine is selected from the group consisting of IL-12, IL-17, IFN- ⁇ , TNF- ⁇ , and IL-23.
  • cytokine is selected from the group consisting of IL-4, IL-10 and TGF- ⁇ .
  • a pharmaceutical composition comprising a polypeptide from about 6 to about 35 amino acid residues, the polypeptide comprising 1 to 3 tetrapeptides having the sequence of PEXP, wherein
  • cell penetrating peptide is selected from the group consisting of Penetratin, Pep-1, Pep-2, VP22, pVEC, pISL, hCT derived peptide, LL-37, Mouse PrP, Transportan, TP10, Arg11, MAP, MPG, KALA, ppTG1, and ppTG20.
  • cytokine is selected from the group consisting of IL-12, IL-17, IFN- ⁇ , TNF- ⁇ , and IL-23.
  • cytokine is selected from the group consisting of IL-4, IL-10 and TGF- ⁇ .
  • composition of any one of clauses 36 to 63, wherein the composition upregulates a Th-2 specific transcriptional factor.
  • a method of treating multiple sclerosis comprising the step of administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition of any one of clauses 1 to 70.
  • cytokine is selected from the group consisting of IL-12, IL-17, IFN- ⁇ , TNF- ⁇ , and IL-23.
  • cytokine is selected from the group consisting of IL-4, IL-10 and TGF- ⁇ .
  • Th-2 specific transcriptional factor is GATA-3.
  • a pharmaceutical formulation comprising the pharmaceutical composition of any one of clauses 1 to 70.
  • FIG. 1 shows pathways underlying functional influence are reconstructed in MetaCore.
  • the network has 75% of its nodes associated with T cell mediate inflammatory diseases by annotations in MetaCore.
  • GILZ was identified as a “divergence” hub functionally linked to multiple up/downstream target genes differentially expressed in activated CD4+ T cells. Functionally inhibitory and positive links are highlighted in red and green, respectively.
  • FIG. 2 shows GILZ:p65 binding analysis by ELISA.
  • Wells coated with human GILZ protein at increasing concentrations (5-40 ng) were probed with recombinant p65 protein at increasing concentrations (0.3125-40 ng) and detected with anti-human GILZ mAb (A).
  • Human CD4+ T cells were stimulated with PPD in the presence or absence of dexamethasone.
  • ELISA plates coated with recombinant human p65 protein (B) or human GILZ protein (C) were probed with T cell cytoplasmic fractions (B) or nuclear fractions (C).
  • the GILZ:p65 binding was detected with anti-human GILZ mAb (B) or anti-human p65 mAb (C).
  • * p ⁇ 0.05 when compared to cells +PPD.
  • FIG. 3 shows intracellular delivery of GILZ protein.
  • CD4+ T cells were permeabilized and incubated with human GILZ protein alone (B) or in 1:20 (molar concentration) GILZ:chariot peptide pre-formed complex (C) or left untreated (A). Intracellular delivery of GILZ protein was detected with fluorescently labeled anti-human GILZ mAb.
  • FIG. 4 shows exogenous GILZ suppressed Th1 cytokines.
  • FIG. 5 shows treatment with rGILZ or wild type GILZ-peptide (GILZ-P) suppresses cytokine secretion by in-vivo primed T cells.
  • CD4+ splenocytes isolated 10 days post-immunization from SJL mice induced EAE were co-cultured with irradiated syngenic APC and restimulated with PLP (40 ⁇ g/ml) in the presence of complex of chariot peptide with rGILZ protein/dexamethasone/GILZ-P at indicated concentrations.
  • FIG. 6 shows treatment with GILZ-peptide (GILZ-P) (SEQ ID NO: 6) suppresses proliferative responses and cytokine secretion by antigen primed T cells.
  • FIG. 6A shows single cell suspensions of CD4 + LNC (5 ⁇ 10 5 cells/well) isolated from SJL mice 10 days post-PLP immunization were co-cultured with irradiated syngenic APC and restimulated with PLP (40 ⁇ g/ml) for a total of 72 h (including an 18-h pulse with [ 3 H]-thymidine) in the presence of indicated concentrations of dexamethasone (10 7 M)/GILZ peptide (125-500 ⁇ M)/control peptide(C-P)(500 ⁇ M)/r-GILZ (100-200 ng)/control-Ig (C-Ig) as shown.
  • FIGS. 6B-D shows CD4 + LNC from PLP-primed mice were restimulated with the immunogen in the presence of r-GILZ/GILZ peptide/control peptide/control Ig at indicated concentration. Supernatants collected 48 h later were assessed for IL-12 (B), IFN- ⁇ (C) and IL-10 (D) by ELISA. Data are presented as mean+/ ⁇ SD., # and @ p ⁇ 0.05 as compared with the cells treated with PLP alone or cells stimulated in the presence of control peptide/control Ig respectively.
  • FIG. 7 shows GILZ peptide treatment inhibits R-EAE.
  • SJL/J mice were immunized with PLP 139-151 in CFA and injected pertussis toxin i.p. on days 0 and 2. Groups of mice received i.p. injection of GILZ-peptide (500 ⁇ g) either on day 0 or on day12/r-GILZ (2 ng)/control peptide (500 ⁇ g) in complex with Pep-1 (0.3 ⁇ M) or Pep-1 alone or vehicle (PBS) on the day of immunization.
  • the severity of EAE is depicted as the mean score per day, which is the cumulative score for each animal divided by the number of days that animal, was observed.
  • FIG. 8 shows that GILZ peptide treatment reduces proinflammatory cytokine responses in R-EAE.
  • CD4+LNC collected 45 days post-immunization from SJL/J mice induced R-EAE and treated with GILZ peptide (GILZ-P) (day0/day12)/r-GILZ/control peptide/vehicle on the day of immunization were cultured with PLP 139-151 (40 ⁇ g/ml)/MBP 87-99 40 ⁇ g/ml/both using syngenic irradiated splenocytes as APC.
  • FIG. 9 shows GILZ-p65 binding analysis.
  • CD4+ cells from the peripheral blood mononuclear cells of individuals vaccinated with the BCG vaccine were stimulated with PPD in the presence or absence of dexamethasone.
  • Nuclear and cytoplasmic extracts were obtained from cells harvested after 24 h. Binding between the plate-bound r-p65 and the cytoplasmic GILZ (A) or the plate-bound r-GILZ and nuclear p65 (B) were detected by anti-GILZ (A) or anti-p65 (B) mAb, respectively.
  • FIG. 1 shows wells coated with r-GILZ at increasing concentrations (5-40 ⁇ M) were probed with full-length r-p65:DDK at increasing concentrations (0.3125-40 ⁇ M) and detected with anti-human GILZ mAb (D).
  • FIG. 10 (A)-(D) shows intracellular delivery of r-GILZ by Pep-1:Jurkat T cells in serum-free HL-1 medium were overlaid with a pre-formed complex of r-GILZ (DDK tag) and Pep-1 at varying concentrations and incubated in a humidified chamber for 1 h at 37° C. Intracellular delivery of r-GILZ was detected using phosphatidylethanolamine-labeled anti-DDK mAb in permeabilized cells and measured by FACS Calibur. Representative images of cells incubated with (A) r-GILZ alone, (C) 20:1 ratio of Pep-1 to r-GILZ, and (B) pep-1 alone are shown.
  • CD4+ splenocytes (5 ⁇ 10 5 cells/well) isolated from antigen-primed SJL mice were co-cultured with irradiated syngenic splenocytes as APC and restimulated with PLP139-151 (40 ⁇ g/ml) for a total of 72 h (including an 18-h pulse with [3H]thymidine) in the presence of dexamethasone (20-7/10 ⁇ 7 M), r-GILZ (100 and 200 ng), GILZ-P (125-500 ⁇ M), and control peptide (C-P)(500 ⁇ M) as shown.
  • dexamethasone (20-7/10 ⁇ 7 M
  • r-GILZ 100 and 200 ng
  • GILZ-P 125-500 ⁇ M
  • C-P control peptide
  • (E) shows data represent mean ⁇ cpm (cpm of the antigen-stimulated cells—cpm of cells only) ⁇ S.D. from three different experiments.
  • (F)-(I) show supernatants collected at 48 h were assessed for IFN- ⁇ (F), IL-12 (G), IL-17 (H), and IL-10 (I) by ELISA. Data are presented as mean ⁇ S.D.
  • (J) shows in separate experiments, draining LNC cultured similarly were harvested at the end of 24 h. Five micrograms of nuclear extracts were tested for binding of the activated p65 NF- ⁇ B subunit to an NF- ⁇ B consensus sequence using the TransAM NF- ⁇ B ELISA kit.
  • the p65 DNA binding activity was calculated as the ratio of absorbance from PLP139-151-stimulated cells to that of unstimulated cells. Values are the average ⁇ S.D. performed 3 times in duplicate.
  • (K)-(L) show real time PCR for T-bet (K) and GATA-3 (L) was performed using 50 ng of cDNA isolated from CD4+LNC of R-EAE mice re-stimulated in vitro with PLP139-151 under the indicated conditions.
  • FIG. 11 shows GILZ-peptide treatment inhibits R-EAE.
  • SJL/J mice induced with R-EAE were administered intraperitoneally GILZ-peptide (500 ⁇ g) either on day 0 or 12 for r-GILZ (2 ng) and control peptide (500 ⁇ g) in complex with Pep-1 (0.3 ⁇ M) or Pep-1 alone, or vehicle (PBS) on the day of immunization.
  • A shows the mean clinical score per day per group.
  • (B) shows the severity of EAE is depicted as the mean score per day, which is the cumulative score for each animal divided by the number of days that animal was observed. Data represent average ⁇ S.D. from two experiments (n 9/group).
  • CD4+ splenocytes isolated 45 days post-immunization from the draining lymph nodes of SJL/J mice induced with R-EAE and treated with GILZ-peptide (500 ⁇ g) either on day 0 or 12 for r-GILZ, control peptide, or vehicle (PBS) were restimulated in vitro with PLP139-151 (40 ⁇ g/ml), MBP87-99 (40 ⁇ g/ml), or both using syngenic irradiated splenocytes as APC for a total of 72 h (including an 18-h pulse with [3H]thymidine).
  • proliferative responses of CD4+ splenocytes are plotted as ⁇ cpm ⁇ S.E. #, p ⁇ 0.05 as compared with the responses of cells from vehicle-treated mice.
  • Supernatant collected at 48 h from separate CD4+ splenocyte cultures were assessed for (D) IL-12, (E) IFN- ⁇ , (F) IL-17, (G) IL-10, and (H) TGF- ⁇ by ELISA.
  • * and # represent p ⁇ 0.05 as compared with vehicle or control peptide-treated groups, respectively.
  • FIG. 12 shows GILZ-peptide treatment suppresses Th1 and enhances Th2 transcription factors in R-EAE.
  • FIG. 13 shows the CD spectrum of GILZ-P at various temperatures and in 5M CaCl 2 .
  • FIG. 14 shows GILZ-peptide treatment inhibits R-EAE.
  • A shows the mean clinical score per day per group.
  • B shows the severity of EAE is depicted as the mean score per day, which is the cumulative score for each animal divided by the number of days that animal was observed.
  • FIG. 15 shows assays of specific cytokines ((A) IFN- ⁇ , (B) TNF- ⁇ , and (C) IL-17) assayed from supernatants collected at 48 hours.
  • a pharmaceutical composition comprising a polypeptide from about 6 to about 35 amino acid residues.
  • the term “about” means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon its particular use under study, and can be readily appreciated by one of skill in the art.
  • the polypeptide comprises 1 to 3 tetrapeptides having the sequence of PXXP, wherein P is proline, and X is any amino acid.
  • a pharmaceutical composition comprising a polypeptide from about 6 to about 35 amino acid residues.
  • the polypeptide comprises 1 to 3 tetrapeptides having the sequence of PEXP, wherein P is proline, E is glutamic acid, and X is any amino acid.
  • the pharmaceutical composition comprises a polypeptide wherein at least one tetrapeptide comprises the amino acid sequence of PEEP (SEQ ID NO: 3). In other embodiments described herein, the pharmaceutical composition comprises a polypeptide wherein at least one tetrapeptide comprises the amino acid sequence of PESP (SEQ ID NO: 4). In yet other embodiments described herein, the pharmaceutical composition comprises a polypeptide wherein at least one tetrapeptide comprises the amino acid sequence of PEAP (SEQ ID NO: 5).
  • the pharmaceutical composition comprises a polypeptide comprising the amino acid sequence of CLSPEEPAPESPQVPEAPGGSAV (SEQ ID NO: 6). In other embodiments described herein, the pharmaceutical composition comprises a polypeptide comprising the amino acid sequence of CSLPEEPAPEAPETPETPEAPGGSAV (SEQ ID NO: 7).
  • the pharmaceutical composition comprises a polypeptide from about 6 to about 35 amino acid residues. In other embodiments described herein, the pharmaceutical composition comprises a polypeptide from about 6 to about 30 amino acid residues. In yet other embodiments described herein, the pharmaceutical composition comprises a polypeptide from about 10 to about 30 amino acid residues. In one illustrative embodiment, the pharmaceutical composition comprises a polypeptide from about 6 to about 22 amino acid residues. In another illustrative embodiment, the pharmaceutical composition comprises a polypeptide from about 12 to about 30 amino acid residues. In another illustrative embodiment, the pharmaceutical composition comprises a polypeptide from about 12 to about 22 amino acid residues.
  • the pharmaceutical composition comprises a polypeptide wherein the polypeptide comprises a polyproline helical conformation.
  • polyproline helical conformation means a helical conformation of a peptide that is commonly associated with more than one proline residues.
  • the pharmaceutical composition further comprises a cell penetrating peptide (also known as a chariot peptide).
  • Chariot and cell penetrating peptides are known by those of skill in the art. For review, see El-Andaloussi et al. (2005) Current Pharm. Design 11:3597-3611 and Wagstaff & Jans (2006) Current Med. Chem. 13:1371-1387.
  • the cell penetrating peptide is selected from the group consisting of Penetratin, Pep-1, Pep-2, VP22, pVEC, pISL, hCT derived peptide, LL-37, Mouse PrP, Transportan, TP10, Arg11, MAP, MPG, KALA, ppTG1, and ppTG20.
  • the cell penetrating peptide is a bipartite peptide (K16ApoE) comprising a polylysine segment linked with the apolipoprotein-E peptide.
  • the cell penetrating peptide is Pep-1.
  • the pharmaceutical composition comprises a polypeptide that is protected from aminopeptidases and/or carbozypeptidases.
  • the polypeptide is amidated at the amino terminus and acetylated at the carboxy terminus.
  • the polypeptide can be modified by the inclusion of one or more conservative amino acid substitutions.
  • conservative amino acid substitutions As is well known to those skilled in the art, altering any non-critical amino acid of a peptide by conservative substitution should not significantly alter the activity of that peptide because the side-chain of the replacement amino acid should be able to form similar bonds and contacts to the side chain of the amino acid which has been replaced.
  • Non-conservative substitutions are possible provided that these do not excessively affect the p65 sequestering activity of the polypeptide and/or reduce its effectiveness in suppressing inflammatory cytokines.
  • a “conservative substitution” of an amino acid or a “conservative substitution variant” of a peptide refers to an amino acid substitution which maintains: 1) the secondary structure of the peptide; 2) the charge or hydrophobicity of the amino acid; and 3) the bulkiness of the side chain or any one or more of these characteristics.
  • hydrophilic residues relate to serine or threonine.
  • Hydrodrophobic residues refer to leucine, isoleucine, phenylalanine, valine or alanine, or the like.
  • “Positively charged residues” relate to lysine, arginine, ornithine, or histidine. “Negatively charged residues” refer to aspartic acid or glutamic acid. Residues having “bulky side chains” refer to phenylalanine, tryptophan or tyrosine, or the like. A list of illustrative conservative amino acid substitutions is given in TABLE 1.
  • the pharmaceutical composition suppresses p65 binding. In other embodiments described herein, the pharmaceutical composition suppresses p65 activation. In yet other embodiments described herein, the pharmaceutical composition inhibits NF- ⁇ B translocation to the nucleus of a cell.
  • the pharmaceutical composition suppresses T cell response. In other embodiments described herein, the pharmaceutical composition suppresses T-bet transcription.
  • the pharmaceutical composition suppresses a pro-inflammatory cytokine. In other embodiments described herein, the pharmaceutical composition suppresses a cytokine associated with Th-1 response.
  • the cytokine is selected from the group consisting of IL-12, IL-17, IFN- ⁇ , TNF- ⁇ , and IL-23.
  • the cytokine is IL-12. In another illustrative embodiment, the cytokine is IL-17. In yet another illustrative embodiment, the cytokine is IFN- ⁇ . In another illustrative embodiment, the cytokine is TNF- ⁇ . In another illustrative embodiment, the cytokine is IL-23.
  • the pharmaceutical composition enhances a cytokine associated with Th-2 response.
  • the cytokine is selected from the group consisting of IL-4, IL-10 and TGF- ⁇ .
  • the cytokine is IL-4.
  • the cytokine is IL-10.
  • the cytokine is TGF- ⁇ .
  • the pharmaceutical composition upregulates a Th-2 specific transcriptional factor.
  • the Th-2 specific transcriptional factor is STAT-6.
  • the Th-2 specific transcriptional factor is GATA-3.
  • the pharmaceutical composition is associated with a Th-2 bias in the Th-1/Th-2 balance.
  • the pharmaceutical composition is for use in the treatment of multiple sclerosis. In some embodiments described herein, the pharmaceutical composition reduces a symptom associated with multiple sclerosis. In one illustrative embodiment, the symptom associated with multiple sclerosis is an inflammatory symptom.
  • a method of treating MS comprises the step of administering to a patient in need thereof a therapeutically effective amount of any of the pharmaceutical compositions described herein.
  • treatment of MS includes, but is not limited to, reduction or prevention of inflammation, inhibition of tissue damage, mitigation of clinical symptoms, and/or promotion of recovery following an acute episode.
  • EAE Experimental autoimmune encephalomyelitis
  • GILZ mimetic polypeptides may inhibit the activity of NF ⁇ B and thereby suppress inflammatory cytokines.
  • the inhibitory potential of the polypeptides is assessed by measuring the functional response of CD4+ T cells from mice induced with EAE to re-stimulation with the priming antigen in vitro in the presence/absence of GILZ/GILZ mimetics.
  • the administration according to the described methods is an injection.
  • the injection is selected from the group consisting of intraarticular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous injections.
  • the injection is an intravenous injection.
  • the administration according to the described methods is performed as a single dose administration. In other embodiments, the administration according to the described methods is performed as a multiple dose administration.
  • pharmaceutical formulations are provided.
  • the pharmaceutical formulation comprises any of the pharmaceutical compositions described herein.
  • the previously described embodiments of the pharmaceutical compositions are applicable to the pharmaceutical formulations described herein.
  • the type of formulation employed for the administration of the compounds may be dictated by the particular compounds employed, the type of pharmacokinetic profile desired from the route of administration and the compound(s), and the state of the patient.
  • the polypeptides may be formulated as pharmaceutical compositions for systemic administration. Such pharmaceutical compositions and processes for making the same are known in the art for both humans and non-human mammals. See, e.g., Remington: The Science and Practice of Pharmacy, (A. Gennaro, et al., eds., 19 th ed., Mack Publishing Co., 1995).
  • the pharmaceutical formulations described herein further comprise a pharmaceutically acceptable carrier.
  • the pharmaceutical formulations described herein further comprise a pharmaceutically acceptable diluent.
  • Diluent or carrier ingredients used in the pharmaceutical compositions containing polypeptides can be selected so that they do not diminish the desired effects of the polypeptide.
  • suitable dosage forms include aqueous solutions of the polypeptides, for example, a solution in isotonic saline, 5% glucose or other well-known pharmaceutically acceptable liquid carriers such as alcohols, glycols, esters and amides.
  • carrier refers to any ingredient other than the active component(s) in a formulation.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. (1985)). The choice of carrier will to a large extent depend on factors such as the particular mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form.
  • the carrier is a liquid carrier.
  • a “patient” as referred to herein can be a human patient or a veterinary patient, such as a domesticated animal (e.g., a pet).
  • the pharmaceutical formulations described herein optionally include one or more other therapeutic ingredients.
  • active ingredient or “therapeutic ingredient” refers to a therapeutically active compound, as well as any prodrugs thereof and pharmaceutically acceptable salts, hydrates, and solvates of the compound and the prodrugs.
  • Other active ingredients may be combined with the described polypeptides and may be either administered separately or in the same pharmaceutical formulation. The amount of other active ingredients to be given may be readily determined by one skilled in the art based upon therapy with described polypeptides.
  • the pharmaceutical formulations described herein are a single unit dose.
  • the term “unit dose” is a discrete amount of the composition comprising a predetermined amount of the described polypeptides.
  • the amount of the described polypeptides is generally equal to the dosage of the described polypeptides which would be administered to an animal or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • parenteral formulations may be suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • a suitable vehicle such as sterile, pyrogen-free water.
  • the preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art.
  • aqueous preparations according to the invention can be used to produce lyophilisates by conventional lyophilization or powders.
  • the preparations according to the invention are obtained again by dissolving the lyophilisates in water or other aqueous solutions.
  • lyophilization also known as freeze-drying, is a commonly employed technique for presenting proteins which serves to remove water from the protein preparation of interest. Lyophilization is a process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient may be included in pre-lyophilized formulations to enhance stability during the freeze-drying process and/or to improve stability of the lyophilized product upon storage. For example, see Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawa et al., Pharm. Res., 8(3):285-291 (1991).
  • solubility of the polypeptides used in the preparation of a parenteral formulation may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing agents.
  • formulations for parenteral administration may be formulated to be for immediate and/or modified release.
  • Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations.
  • a polypeptide may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • the formulations can also be presented in syringes, such as prefilled syringes.
  • the dosages of the polypeptides can vary significantly depending on the patient condition and the severity of the disease to be treated.
  • the effective amount to be administered to a patient is based on body surface area, patient weight or mass, and physician assessment of patient condition.
  • Suitable dosages of the polypeptides can be determined by standard methods, for example by establishing dose-response curves in laboratory animal models or in humans in clinical trials.
  • suitable dosages of polypeptides include from about 1 pg/kg to about 10 ⁇ g/kg, from about 1 pg/kg to about 1 ⁇ g/kg, from about 100 pg/kg to about 500 ng/kg, from about 1 pg/kg to about 1 ng/kg, from about 1 pg/kg to about 500 pg/kg, from about 100 pg/kg to about 500 ng/kg, from about 100 pg/kg to about 100 ng/kg, from about 1 ng/kg to about 10 mg/kg, from about 1 ng/kg to 1 mg/kg, from about 1 ng/kg to about 1 ⁇ g/kg, from about 1 ng/kg to about 500 ng/kg, from about 100 ng/kg to about to about
  • GILZ mimetics are designed based on the primary structure of GILZ, a glucocorticoid-inducible gene, and its interaction with the p65 subunit of NF- ⁇ B.
  • a cost-effective hybrid approach that combines initial in-silico screening with subsequent cellular validation is used to identify functional GILZ mimetics. Since activated p65 is present only in stimulated cells treatment with GILZ mimetics is less likely to cause global immunosuppression.
  • the GILZ mimetics may circumvent the complications associated with the negative effects of glucocorticoid receptor mediated transactivation and transrepression of multiple genes that follows synthetic glucocorticoid treatment by sequestering the active p65 within the cytoplasm.
  • GEO gene expression omnibus
  • Bioinformatic analyses were performed using the MetaCoreTM software package (http://portal.genego.com). Comparative analyses after normalization of differentially expressed genes showed that while both anergic T cells and dexamethasone treated T cells expressed GILZ, it was significantly upregulated in the later cells. Enrichment of functional processes was calculated by “GeneGo process networks” using a threshold of 0.001 and a false discovery rate ⁇ 5%. Although GILZ was present in anergic T cells and in dexamethasone treated cells, its expression level did not reach the threshold set for statistical significance in the enrichment analysis. Hence GILZ was added manually to the immune response processes prior to building networks.
  • GILZ was identified as a “divergence” hub functionally linked to multiple genes differentially expressed in activated CD4+ T cells. Interactome analysis revealed that while the pro-inflammatory factors T-bet, IFN- ⁇ and TNF- ⁇ inhibit GILZ; anti-inflammatory factors GATA-3 and IL-13 are activated by GILZ (see FIG. 1 ). GILZ mediated inhibition of anti-apoptotic factor, Bcl-XL, may indicate a potential role for GILZ in the deletion of activated T cells. Since many of these mechanisms occur following NF- ⁇ B transactivation, an important role played by GILZ may reside in its ability to prevent nuclear translocation of the p65 subunit of NF-K ⁇ (see FIG. 1 ).
  • GILZ The primary sequence of GILZ is homologous with that of the evolutionarily conserved TGF ⁇ -stimulated clone 22 gene (TSC-22) and porcine delta sleep inducing peptide (DSIP). They share the leucine-zipper (LZ) motif commonly found in transcriptional regulatory proteins and a proline rich carboxy (COOH) terminus. Solution structure suggested that while the LZ motif of DSIP adopts helical conformation, the proline rich COOH region is more flexible. This is consistent with the observations that the LZ domains facilitate dimerization and the flexible proline rich regions participate in protein-protein interactions.
  • TSC-22 evolutionarily conserved TGF ⁇ -stimulated clone 22 gene
  • DSIP porcine delta sleep inducing peptide
  • p65 possesses a rel homology domain (RHD), a nuclear localization sequence (NLS) masked in resting cells by the I ⁇ B inhibitory proteins and carboxy terminal transactivation domains (TAD).
  • the TAD-1 (residues 521-551) is the dominant contributor to the transactivation activity of p65.
  • proline rich human GILZ-COOH is identical with that of the TSC22 and the DSIP.
  • Homology models of GILZ mimetics are built using the solution structure of DSIP (PDB: 1DIP) as the template by modeling programs (e.g. Geno 3D/SWISS MODEL). The structure of the predicted models is refined by energy minimization and removal of unfavorable bond geometry.
  • the GILZ mimetics may retain a structural representation with reference to the adjacent residues and adopt a similar conformation as wild type GILZ.
  • GILZ-mimetics are superimposed over the carboxy terminal region of the predicted GLIZ model and PDB: 1DIP. GILZ mimetics within 5 ⁇ RMSD when superimposed over both are selected for in silico docking NMR studies suggest that the 133 residue TAD of p65 adopts ⁇ -helical conformation facilitated by the Asp/Glu rich domain.
  • a structure homology search revealed similarity between the acidic activation domain of the elongation factor eEF3 (PDB: 3H7H) and the TAD of p65.
  • repositories in the public domain include multiple models of human p65 generated by homology-modeling.
  • GILZ mimetics are used to evaluate the p65 binding potential of GILZ mimetics using a soft docking algorithm implemented in BiGGER (Biomolecular Complex Generation with Global Evaluation and Ranking).
  • the coordinates of GILZ mimetics are systematically rotated in discrete steps and translated against the surface of human p65 TAD. In general two proteins are considered in contact with each other if the distance between the C ⁇ atoms is ⁇ 5 ⁇ .
  • Top ranked GILZ mimetics within 5 ⁇ distance of the residues critical for transactivation of p65 are selected for further analysis.
  • Selected GILZ mimetics may be synthesized as peptide amides and amino terminal acetylation to minimize charge-helix dipole interaction and enhance helical stability.
  • Proline rich regions in polypeptides involved in transient protein-protein interactions often assume an extended helical conformation.
  • the secondary structure and the binding kinetics between the GILZ protein/GILZ mimetics and the p65 protein are determined by circular dichroism and BIAcore analysis, respectively (Srinivasan et al. (2001) J Immunol. 167(1):578-85; Srinivasan et al., (2005) J Biol Chem. 280(11):10149-55).
  • Circular dichroism (CD) measurements of the synthesized GILZ mimetics are recorded to demonstrate the secondary structure of GILZ mimetics in solution.
  • Mimetics with defined extended helical conformation may be chosen for further functional characterization.
  • the direct interaction between the purified human GILZ and the purified or cellular p65 protein was studied using a modified ELISA.
  • the human GILZ-GST protein and the anti-human GILZ mAb were purchased from Abnova, Teipie City, Taiwan.
  • Purified human p65 protein and the anti-human p65 mAb were from Active Motif, Carlsbad Calif.
  • PBMC Peripheral Blood Mononuclear Cells
  • PBMC isolated from 10 cc blood of a healthy donor [vaccinated with the Mycobacterium bovis bacille Calmette-Guerin (BCG)] were cultured in complete RPMI at 1 ⁇ 105 cells/well and stimulated with purified protein derivative [PPD:10TU (tuberculin units)/ml] in the presence or absence dexamethasone (100 ⁇ m/ml).
  • CD4+ T cells were isolated 24 and 48 hrs later by magnetic separation and cytoplasmic and nuclear fractions extracted for binding analysis. The protocol was approved by the Indiana University Human Studies Committee, Institutional Review Board.
  • ELISA plates were coated with increasing concentrations of the GILZ protein and probed with increasing concentrations of the p65 protein followed by detection with anti-human GILZ mAb.
  • ELISA plates were coated with the p65 protein (20 ng), and probed with the cytoplasmic fraction from T cells. Binding of the plate bound p65 with the cytoplasmic GILZ protein was detected with anti-human GILZ mAb.
  • wells coated with the GILZ-GST protein (20 ng) were probed with the nuclear fractions from T cells and the GILZ:nuclear p65 binding was detected with anti-human p65 mAb. The bound complex was detected with TNB at absorbance of 450 nm.
  • the GILZ:anti-GILZ complex formation was inhibited by p65 as evidenced by the decreasing absorbance with increasing concentration of the p65 protein (see FIG. 2A ).
  • the interaction appears to be non-linear with optimal binding at 1:4 ratio of p65 to GILZ proteins.
  • Absorbance over the background was not observed in wells coated with anti-human GILZ mAb, probed with purified p65 and detected with anti-human p65 mAb. The absorbance was highest in wells probed with anti-GILZ mAb alone.
  • T cell activation decreases GILZ expression
  • treatment of activated T cells with dexamethasone upregulates GILZ expression. Consistent with this increased absorbance in p65 coated wells probed with the cytoplasmic fraction of antigen activated CD4+ T cells treated with dexamethasone as compared with that from untreated cells was observed (see FIG. 2B ).
  • Activated T cells express increased p65 in the nucleus. Consistently, the absorbance was significantly decreased in wells coated with purified human GILZ protein and probed with the nuclear fraction of antigen stimulated cells treated with dexamethasone as compared to that from untreated cells (see FIG. 2C ).
  • BIAcore experiments are performed.
  • BIAcore measures surface plasmon resonance (SPR) for real-time monitoring of intermolecular interactions.
  • Functional GILZ-mimetics may bind the p65 protein with similar kinetics as wild type GILZ. Both direct and competitive kinetics for the interactions between the human GILZ protein/select GILZ mimetics and the human p65 protein are determined.
  • Purified p65 protein is immobilized on a CM-5 sensor chip using the amine coupling method as described.
  • Human GILZ protein or GILZ mimetics at varying concentrations are injected as analytes. The binding is monitored in real time in terms of response units and the p65 surface regenerated between injections.
  • anti-human p65 or purified human GILZ protein are immobilized on the CM-5 sensor chip.
  • Mixtures of purified human p65 protein with increasing concentrations of each GILZ mimetic are injected as analytes.
  • the binding is monitored in real time in terms of response units and the bound protein regenerated between injections by washing with 5 mM NaOH.
  • Data from the sensograms is analyzed to calculate the binding kinetics and affinity constant for the interaction using BIAevaluation and BIAsimulation software.
  • GILZ human immunodeficiency virus
  • T cells isolated from PBMC by magnetic cell separation were seeded with irradiated autologous PBMC (T cells depleted) as APC at 1:1.5 ratio in serum-free RPMI (with 5% penicillin/streptomycin), and stimulated with PPD (20 ⁇ m/ml) alone or in the presence of dexamethasone (100 ⁇ m/ml) or 20:1 M complex of chariot peptide with GILZ protein (0.7 ⁇ m/ml)/anti-human GILZ mAb (1 ⁇ m/ml)/anti-human p65 mAb (1 ⁇ m/ml)/control antibody (1 ⁇ m/ml).
  • cytokines including human IFN- ⁇ , TNF- ⁇ , IL-12 p40 and IL-10 using BD OptEIATM kits. Differences in the secretion of cytokines between the groups were determined by one way ANOVA followed by Tukey's post-hoc analysis (see FIG. 4 ).
  • cytokine secretions in PPD stimulated T cells treated with peptide carrier alone/peptide carrier and non specific immunoglobulin did not vary significantly from that of PPD stimulated cells. Cytokine secretion was not significant in unstimulated cell cultures in all conditions.
  • NF- ⁇ B is integrally involved in the transcriptional upregulation of multiple proinflammatory cytokines including IFN- ⁇ and IL-12, that mediate tissue destruction in MS.
  • Exogenous GILZ has been shown to suppress Th1 cytokines
  • the ability of GILZ mimetics to inhibit NF- ⁇ B translocation and suppress T cell responses is assessed in EAE, a well established model for MS.
  • mice Relapsing remitting EAE is induced in 8-10-week old female SJL/J mice.
  • the mice were immunized with 100 ⁇ g of PLP 139-151 in PBS emulsified 1:1 in complete Freund's adjuvant supplemented with 200 ⁇ g of Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, Mich.) distributed over four sites on the lateral hind flanks subcutaneously.
  • Mice were injected with 100 ng of pertussis toxin (List Biological Laboratories, Campbell, Calif.) intraperitonealy on the day of immunization and 2 days later (Srinivasan et al., (2002) J Immunol, 169(4):2180-8).
  • Proliferative responses of CD4+ lymph node cells and splenocytes harvested 10 days post-induction upon re-stimulation in vitro with PLP 139-151 in the presence or absence of chariot peptide complexed with purified GILZ protein and/or GILZ mimetics at varying concentrations is determined as described.
  • IL-2, TNF- ⁇ , IFN- ⁇ , IL12-p40, IL-17, IL-4, IL-5, IL-10 and TGF- ⁇ ) of the cells following similar in vitro restimulation is determined by ELISA.
  • Measurement of relative quantities of Th1 and Th2 specific transcription factors is determined by real time PCR using ABI primers.
  • Nuclear and cytoplasmic protein fractions of CD4+ T cells obtained using a nuclear extraction kit are assessed for NF- ⁇ B activation and p65 nuclear translocation using Trans AM NF- ⁇ B kits (Active Motif, Carlsbad, Calif.).
  • the effect of exogenous GILZ and GILZ mimetics on phosphorylation and proteolytic degradation of I ⁇ B proteins is determined by Western blot analysis of nuclear and cytoplasmic protein fractions from CD4+ T cells pretreated with GILZ/chariot peptide complex prior to stimulation.
  • a peptide derived from the TAD1 of p65 has been shown to suppress TNF- ⁇ induced NF- ⁇ B activation by preventing nuclear translocation of p65.
  • GILZ has been shown to bind p65 in the cellular cytoplasm and inhibit its nuclear translocation. Human GILZ and the mouse GILZ share identical proline rich regions. Treatment with GILZ mimetics may inhibit NF- ⁇ B activation, T-bet transcription, and reduce pro-inflammatory cytokine secretion (see FIG. 5 ) to suppress antigen activated T cells in EAE. Activated NF- ⁇ B is expected to be lower in cells treated with exogenous GILZ. Multiple reports suggest that the NF- ⁇ B mediates suppression of apoptosis. Exogenous GILZ and GILZ mimetics may suppress NF- ⁇ B translocation and hence increase apoptosis of activated CD4+ T cells.
  • dexamethasone/GILZ peptide/recombinant GILZ protein (r-GILZ) (Abnova, Teipie City, Taiwan)/control peptide was incubated with the amphipathic chariot peptide (Pep-1) at 1:20 M ratio for 20 mins at room temperature before adding to the cell cultures (Deshayes et al., (2010) Biochim Biophys Acta, 1798(12):2304-14).
  • CD4+ lymph node cells isolated from the draining lymph nodes harvested from SJL/J mice 10 days after immunization with PLP 139-151 were cultured in 96-well plates at 5 ⁇ 10 6 cells/well in complete RPMI and restimulated with the PLP peptide (40 ⁇ g/ml) for 72 h, including a final 16-h pulse with [ 3 H]-thymidine.
  • Cultures contained dexamethasone (10 7 M)/different concentrations of GILZ peptide (125 ⁇ M-500 ⁇ M)/r-GILZ (100 ng & 200 ng)//Pep-1 (0.3 ⁇ M) in triplicate wells.
  • mice were administered intraperitonealy r-GILZ (2 ng/mouse) (Abnova, Teipie City, Taiwan) or GILZ peptide (GILZ-P) (SEQ ID NO: 6) or control peptide (500 ⁇ g/mouse) mixed with Pep1 (0.3 ⁇ M/mouse) in 100 ⁇ l PBS on the day of immunization.
  • GILZ-P GILZ peptide
  • Pep1 0.3 ⁇ M/mouse
  • GILZ-P SEQ ID NO: 6
  • SJL/J mice immunized with PLP 139-151 were administered vehicle or 500 ⁇ g of the GILZ peptide/control peptide/2 ng of r-GILZ/Pep-1 alone on the day of EAE induction or 500 ⁇ g of the GILZ peptide 12 days post-induction.
  • the average clinical score per day was significantly lower in mice treated with GILZ-peptide (day 0/day12)/r-GILZ as compared with the control groups (see FIG.
  • mice treated with GILZ-peptide on day 0/day12 (mean clinical score: 0.56+/ ⁇ 0.21) and 1.78+/ ⁇ 1.01 respectively) or r-GILZ (mean clinical score 0.17+/ ⁇ 0.14) as compared with the vehicle treated mice (mean clinical score 6.25+/ ⁇ 2.9) or the control peptide or Pep-1 treated mice (mean clinical score 5.7+/ ⁇ 3.4 and 4.7+/ ⁇ 0.8 respectively) (see FIG. 7B ).
  • mice that received GILZ-peptide (day 0/day 12) or r-GILZ exhibited no relapse and continued to be protected for the entire period of observation (see FIG. 7A ).
  • GILZ peptide treatment mediates skewing of Th1 to Th2 cytokine response in R-EAE.
  • the effect of the GILZ peptide on T cell cytokine secretion was evaluated. Since the PLP 139-151 induced R-EAE in SJL/J mice follows a relapsing remitting course secondary to epitope spreading and since the mice treated with GILZ peptide or r-GILZ were protected against disease relapse, the response of T-cells to PLP 139-151 and to an immunodominant epitope of another encephalitogenic antigen MBP were evaluated.
  • LNC isolated 45 days post-immunization from mice induced with R-EAE and treated with the GILZ peptide (GILZ-P) (SEQ ID NO: 6) (on day 0 or day 12 post-immunization) or r-GILZ protein (SEQ ID NO: 1) secreted significantly reduced Th1 cytokines TNF- ⁇ , IFN- ⁇ and IL-12 in response to either PLP 139-151 or MBP 87-99 as compared with the cells from vehicle or the control peptide treated mice (see FIGS. 8A , 8 B, and 8 C).
  • GILZ-P GILZ peptide
  • SEQ ID NO: 6 secreted significantly reduced Th1 cytokines TNF- ⁇ , IFN- ⁇ and IL-12 in response to either PLP 139-151 or MBP 87-99 as compared with the cells from vehicle or the control peptide treated mice (see FIGS. 8A , 8 B, and 8 C).
  • LNC from R-EAE mice treated with GILZ-peptide/r-GILZ secreted higher IL-10 in response to the PLP 139-151 and/or MBP 87-99 as compared with the vehicle or control peptide treated mice (see FIG. 8D ). No significant cytokine secretion was observed in cultures stimulated with a previously unexposed antigen, ova.
  • GILZ-P 115-137 and a control peptide (control-P) of scrambled residues were synthesized as peptide amides and the PLP 139-151 (HSLGKWLGHPDKF) and MBP 89-97 (VHFFKNIVTPRTP) as peptide acids.
  • the amino-terminal of GILZ-P, control-P, and MBP 89-97 were acetylated. All peptides were 95% pure as confirmed by mass spectrometry.
  • Recombinant human p65 protein (r-p65) and purified r-GILZ with C-terminal DDK and biotinylated anti-DDK antibody were from OriGene Technologies Inc., Rockville, Md.
  • Partial length p65 (p65 ⁇ C14) and anti-p65 mAb were from Active Motif, Carlsbad, Calif.
  • Recombinant mouse GILZ protein and the mouse anti-GILZ mAb were from Abnova Corporation, Walnut, Calif.
  • the mouse anti-GILZ mAb exhibits cross-reactivity with the human GILZ.
  • High binding ELISA plates coated with 40 ⁇ M r-p65/r-GILZ were probed with cytoplasmic/nuclear extracts, respectively, of CD4+ peripheral blood mononuclear cells stimulated with purified protein derivative (10 units/ml) for 48 h in the presence of dexamethasone (100 ⁇ g/ml). Binding of the plate-bound r-p65 with cytoplasmic GILZ and the plate-bound GILZ with nuclear p65 was detected with anti-GILZ mAb or anti-p65 mAb, respectively, followed by trinitrobenzene substrate.
  • r-GILZ For detecting direct interaction of r-GILZ (5-40 ⁇ M), captured wells were probed with r-p65 (0.325-40 ⁇ M) at 22° C. for 2 h and detected with peroxidase-conjugated anti-GILZ mAb followed by trinitrobenzene substrate.
  • Absorbance at 650 nm was measured between 0 and 300 s with a mixing time of 0.30 s and a 5-s interval between readings.
  • mice induced with EAE were administered intraperitoneally with vehicle/Pep1 (0.3 ⁇ M/mouse) mixed with r-GILZ (2 ng/mouse), of GILZ-P, or control-P (500 ⁇ g/mouse) in 100 ⁇ l of PBS on the day of immunization (day 0).
  • vehicle/Pep1 0.3 ⁇ M/mouse
  • r-GILZ 2 ng/mouse
  • control-P 500 ⁇ g/mouse
  • Pep-1-mediated delivery complexes of Pep-1•r-GILZ at varying proportions (50:1 ⁇ M to 1.25:1 ⁇ M or 10:5 ⁇ M in PBS) were incubated at room temperature for 30 min.
  • Jurkat T cells were cultured in complete HL-1 medium supplemented with 5% fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 50 units/ml of penicillin, 50 mg/ml of streptomycin, and 5 ⁇ 10 5 M ⁇ 2-mercaptoethanol in a humidified chamber containing 5% CO 2 at 37° C. for 24 h.
  • the cells were then rested for 2 h, washed, and cultured in a 96-well culture plate at 1 ⁇ 10 4 cells/well in serum-free medium for 60 min. Confluent T cells were then overlaid with the preformed Pep-1•GILZ complexes and incubated at 37° C. for 1 hour. The cells were then extensively washed, permeabilized, and incubated with phycoerythrin-labeled anti-DDK for 30 min at 4° C. Subsequent to washing, the cells were fixed in PBS, 2% paraformaldehyde. The efficiency of r-GILZ intracellular delivery was assessed by measuring the mean fluorescence intensity using FACS Calibur flow cytometer (BD Biosciences).
  • the LNC/splenocytes were harvested 10 and 45 days post-immunization from R-EAE mice administered vehicle, r-GILZ, control-P, or GILZ-P on days 0 and 12 or left untreated.
  • CD4+ cells isolated by microbead separation were cultured in a transwell system with irradiated syngenic splenocytes as APC in complete HL-1 medium and restimulated with 40 ⁇ g/ml of PLP 139-151 , MBP 87-99 or both, and ova in the presence or absence of dexamethasone (10 ⁇ 7 -20 ⁇ 7 M), r-GILZ (100-200 ng), GILZ-P (125-500 ⁇ M), or control-P (500 ⁇ M) preincubated with Pep-1 at a 1:20 M ratio for 20 min with Pep-1 alone.
  • r-p65 To detect direct interaction, the ability of r-p65 to inhibit the binding of anti-GILZ mAb with the plate-bound r-GILZ was assessed. The GILZ anti-GILZ complex formation was inhibited by r-p65 in a dose-dependent manner (see FIG. 9C ). To determine the kinetics of interaction between r-GILZ/GILZ-P and the p65, plate-bound r-GILZ, GILZ-P, and control-P at varying concentrations was probed with full-length r-p65-DDK. The percent of p65 binding decreased with decreasing concentrations of r-GILZ/GILZ-P (see FIG. 9E ).
  • r-GILZ/GILZ-P/control-P were mixed with an amphipathic chariot peptide, Pep-1, which rapidly associates through hydrophobic noncovalent interactions and forms stable nanoparticle complexes in solution independent of cargo sequence or size (see FIG. 10A-10C ).
  • the efficiency of Pep-1 to deliver biological molecules was investigated by incubating Jurkat T cells with preformed complexes of Pep-1 and r-GILZ at varying concentrations. Intracellular delivery was seen in cells incubated with a r-GILZ•Pep-1 complex but not in cells incubated with r-GILZ/Pep-1 alone (see FIG. 10A-10C ).
  • the delivery was most efficient in cells incubated with Pep-1 and r-GILZ at the molar ratio of 50/20:1 as evidenced by the higher mean fluorescence intensity (see FIG. 10D ).
  • the delivery efficiency decreased with decreasing concentrations of Pep-1 (see FIG. 10D ).
  • the mean fluorescence intensity was equivalent in untreated cells and cells overlaid with r-GILZ/Pep-1 alone (see FIG. 10D ).
  • Pep-1 has been effectively used for intracellular delivery of different cargos; including peptide inhibitors of protein kinases, apoptotic protein, and small interfering RNA.
  • the GILZ-P-mediated reduction in proliferation was investigated for its association with the modulation of cytokine response and p65 transactivation.
  • CD4+ LNC from PLP 139-151 -primed mice re-stimulated in vitro in the presence of dexamethasone/GILZ-P/r-GILZ secreted significantly lower pro-inflammatory IFN- ⁇ (see FIG. 10F ), IL-12 (see FIG. 10G ), and IL-17 (see FIG. 10H ) but elevated anti-inflammatory IL-10 cytokine (see FIG. 10I ) as compared with untreated/control-P-treated cells.
  • CD4+ LNC re-stimulated with PLP 139-151 in the presence of dexamethasone/r-GILZ/GILZ-P exhibited significantly decreased T-bet mRNA (see FIG. 10I ), the canonical Th1 transcription factor, and elevated GATA-3 (see FIG. 10L ), the Th2 transcription factor, as compared with unstimulated/control-P-treated cells.
  • significantly decreased p65-specific DNA binding activity was observed in nuclear extracts of CD4+ LNC-restimulated with PLP 139-151 in the presence of dexamethasone/r-GILZ/GILZ-P as compared with untreated or control-P-treated cells (see FIG. 10J ).
  • mice were induced by R-EAE and administered a single intraperitoneal injection of PBS or a complex of Pep-1 and r-GILZ/GILZ-P/control-P. Separate groups of mice received a single dose of Pep-1•GILZ-P on day 12 post-immunization. The average clinical score per day was significantly lower in mice treated with GILZ-P (day 0/day12), r-GILZ as compared with the control groups (see FIGS. 11A and 11B ).
  • mice treated with GILZ-P day 0, 2.25 ⁇ 0.72; day 12, 15.75 ⁇ 4.85) or r-GILZ (7.13 ⁇ 2.8) as compared with vehicle (28.5 ⁇ 5.57) or control-P (33 ⁇ 7.5)-treated mice.
  • vehicle or control-P-treated mice exhibited clinical relapse after initial remission
  • the mice that received GILZ-P/r-GILZ exhibited minimal relapse and continued to be protected for the entire period of observation (see FIG. 11A ).
  • GILZ-Peptide Suppresses T Cell Responses in R-EAE
  • mice The average proliferative response of unstimulated CD4+ splenocytes for vehicle, r-GILZ, GILZ-P (day 0), GILZ-P (day 12), and control-P-treated groups of mice was 2,695 ⁇ 608, 2,037 ⁇ 603, 2,376 ⁇ 602, 1,823 ⁇ 532, and 2,645 ⁇ 425, respectively. No significant difference was observed in the proliferative responses of splenocytes to ovalbumin between the different treatment groups (see FIG. 11C ).
  • IL-12 was significantly lower in CD4+ cells from mice treated with GILZ-P 12 days post-immunization, the reduction in IFN- ⁇ was not significant as compared with that from control groups of mice.
  • the IL-17 secretion by PLP 139-151 re-stimulated CD4+ cells from mice treated on day 0 with r-GILZ (10.66 ⁇ 0.2 pg/ml) or GILZ-P (10.85 ⁇ 0.3 pg/ml) was significantly decreased as compared with cells from vehicle (12 ⁇ 0.7 pg/ml) or control-P (11.95 ⁇ 0.11 pg/ml)-treated mice (see FIG. 11F ).
  • the anti-inflammatory IL-10 secretion was significantly increased in the PLP 139-151 -stimulated cultures of CD4+LNC from mice treated on day 0 with GILZ-P (2316 ⁇ 52.9 pg/ml) as compared with that from vehicle (1456 ⁇ 83.8 pg/ml) or control-P (1488 ⁇ 22.9 pg/ml)-treated mice (see FIG. 11G ).
  • MBP 87-99 but not PLP 139-151 stimulation significantly increased TGF- ⁇ secretion by CD4+ splenocytes from mice treated on day 0 with GILZ-P (33.31 ⁇ 4 pg/ml) or r-GILZ (31.7 ⁇ 0.8 pg/ml) as compared with vehicle (25.3 ⁇ 2.9 pg/ml) or control-P (11.8 ⁇ 4.7 pg/ml)-treated mice (see FIG. 11H ). No significant difference was observed in the response of the CD4+LNC from the different groups of mice to ova, a control antigen.
  • GILZ-Peptide Increases Expression of Regulatory Molecules in T Cells in R-EAE
  • GILZ-peptide The ability of GILZ-peptide to facilitate Th1 to Th2 skewing was evaluated in vivo by modulating specific transcription factors, T-bet and GATA-3, respectively.
  • RT-PCR showed that the CD4+ LNC from mice induced EAE and treated on day 0 with vehicle/r-GILZ/GILZ-P/control-P exhibited significantly lower T-bet but elevated GATA-3 mRNA following re-stimulation with PLP 139-151 (see FIGS. 12A and 12B ).
  • GILZ has been shown to induce IL-10 secretion and promote regulatory T cell differentiation. Accumulation of FOXP3+IL-10 secreting regulatory T cells in the CNS has been associated with disease recovery in EAE.
  • mice treated on day 0 with r-GILZ/GILZ-P exhibited significantly higher expressions of IL-10 and FoxP3 mRNA as compared with that from vehicle/control-P-treated mice (see FIGS. 12C and 12D ).
  • CD measurements can be recorded on a JASCO model, J-710 spectropolarimeter, (Jasco Inc, Easton, Md.) as described previously.
  • the samples can be prepared by dissolving lyophilized GILZ peptide or control peptides at 100 ⁇ M concentration in citrate buffer (1 mM sodium citrate, 1 mM sodium borate, 1 mM sodium phosphate, 15 mM NaCl) with pH adjusted at 7.0.
  • CD spectra can be collected using a 1 cm path-length quartz cuvette at 50° C. or 90° C. in the 190- to 270-nm wavelength range with a 0.5 nm resolution and a scan rate of 200 nm/min.
  • Reported spectra can represent the unsmoothed averages of 30 scans. Each spectrum can be measured three times with individually prepared solutions.
  • Raw CD signals (in millidegrees) can be converted to mean residue molar ellipticity ( ⁇ ) in deg cm 2 /dmol using the formula [ ⁇ ] MRW 5 [ ⁇ ]obs/101cn, where ⁇ obs is the observed ellipticity, 1 is the pathlength in centimeters, c is the molar concentration of peptide, and n is the number of residues in the peptide.
  • ⁇ obs the observed ellipticity
  • 1 is the pathlength in centimeters
  • c is the molar concentration of peptide
  • n is the number of residues in the peptide.
  • CD spectra can be recorded with GILZ-peptide dissolved in 5M CaCl 2 .
  • mice 8-10 wks old SJL/J mice (The Jackson Laboratory, Bar Harbor, Me.) can be immunized with a solution of proteolipin protein peptid (PLP 139-151 ) (100 ⁇ g/mouse) in phosphate buffered saline emulsified with equal volume of complete Freund's adjuvant 200 ⁇ g of Mycobacterium tuberculosis H37RA (Difco Laboratories). Each mouse can receive 25 ul given subcutaneously in the lateral flanks.
  • proteolipin protein peptid PBP 139-151
  • mice can be administered intra-peritoneally vehicle/dexamethasone (dex:0.3 mg/kg)/(500 ⁇ g) of GILZ-P/CP-1/CP-2 mixed with carrier peptide Pep1 (0.304/mouse) in 100 ⁇ l PBS on the day of immunization.
  • a group of mice can receive GILZ-P (100 ⁇ g/day): Pep-1 complex on days 7, 8, 9 and 10 post-induction.
  • CP-1 is a control peptide for the GILZ mimic structure
  • CP-2 is a control peptide for the GILZ mimic amino acid sequence.
  • Mice can be monitored daily and the clinical score will be recorded.
  • the mean clinical score per day per group is shown in FIG. 14(A) .
  • the severity of EAE is depicted in FIG. 14(B) as the mean score per day, which is the cumulative score for each animal divided by the number of days that animals were observed.
  • Peripheral blood from R-EAE mice administered vehicle/dexamethasone/r-GILZ (2 ng/mouse)/(500 ⁇ g) of GILZ-P/CP-1/CP-2/GILZ-P (100 ⁇ g) on days 7, 8, 9 and 10 can be collected from tail vein on day 4 and day 7 post-disease induction.
  • Serum cytokines can be assessed using the OptEIA kits (BD Biosciences, CA).
  • Draining lymph nodes and spleen can be harvested 35 days pot-immunization from R-EAE mice administered vehicle/dexamethasone/(500 ⁇ g) of GILZ-P/CP-1/CP-2/GILZ-P (100 ⁇ g) on days 7, 8, 9 and 10.
  • Single cell suspensions of lymph node cells can be cultured in complete RPMI in the presence or absence of 40 ⁇ g/ml of PLP 139-151 /MBP 87-99 /both/ova.
  • Supernatants collected at 48 hours can be assessed for specific cytokines
  • FIGS. 15(A) , 15 (B), and 15 (C) show the assayed results for IFN- ⁇ , TNF- ⁇ , and IL-17, respectively.

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11183272B2 (en) 2018-12-21 2021-11-23 Biontech Us Inc. Method and systems for prediction of HLA class II-specific epitopes and characterization of CD4+ T cells
US11478526B2 (en) * 2016-02-22 2022-10-25 Indiana University Research And Technology Corp. Methods of reducing neuroinflammation or toxicity induced by amyloid beta (abeta) using glucocorticoid induced leucine zipper (GILZ) analog peptides
US11650211B2 (en) 2017-02-12 2023-05-16 Biontech Us Inc. HLA-based methods and compositions and uses thereof
US11793867B2 (en) 2017-12-18 2023-10-24 Biontech Us Inc. Neoantigens and uses thereof
US12246067B2 (en) 2018-06-19 2025-03-11 Biontech Us Inc. Neoantigens and uses thereof
US12303561B2 (en) 2017-04-03 2025-05-20 Biontech Us Inc. Protein antigens and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040194160A1 (en) 1997-04-28 2004-09-30 Applied Research Systems Ars Holding N.V. Intracellular modulators of apoptopic cell death pathways
WO2007045247A2 (fr) * 2005-10-19 2007-04-26 Enkam Pharmaceuticals A/S Peptides derives d'un recepteur du facteur de croissance des fibroblastes
US20090061462A1 (en) 2003-08-13 2009-03-05 Michelitsch Melissa D Prion-specific peptide reagents
US20100055701A1 (en) 2008-08-27 2010-03-04 Yigang Tong Fusion proteins for imaging the nucleus and chromosomes of live cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040194160A1 (en) 1997-04-28 2004-09-30 Applied Research Systems Ars Holding N.V. Intracellular modulators of apoptopic cell death pathways
US20090061462A1 (en) 2003-08-13 2009-03-05 Michelitsch Melissa D Prion-specific peptide reagents
WO2007045247A2 (fr) * 2005-10-19 2007-04-26 Enkam Pharmaceuticals A/S Peptides derives d'un recepteur du facteur de croissance des fibroblastes
US20100055701A1 (en) 2008-08-27 2010-03-04 Yigang Tong Fusion proteins for imaging the nucleus and chromosomes of live cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Ayroldi, et al. "Glucocorticoid-Induced Leucine Zipper (GILZ); a New Important Mediator of Glucocorticoid Action" The FASEB Journal (2009), vol. 23, p. 3649-3658; p. 3654, co1. 1, paragraph 2, Fig. 1.
PCT International Search Report and Written Opinion completed by the ISA/US on Jul. 31, 2012 and issued in connection with PCT/US2012/026962.

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11478526B2 (en) * 2016-02-22 2022-10-25 Indiana University Research And Technology Corp. Methods of reducing neuroinflammation or toxicity induced by amyloid beta (abeta) using glucocorticoid induced leucine zipper (GILZ) analog peptides
US11650211B2 (en) 2017-02-12 2023-05-16 Biontech Us Inc. HLA-based methods and compositions and uses thereof
US11965892B2 (en) 2017-02-12 2024-04-23 Biontech Us Inc. HLA-based methods and compositions and uses thereof
US12303561B2 (en) 2017-04-03 2025-05-20 Biontech Us Inc. Protein antigens and uses thereof
US11793867B2 (en) 2017-12-18 2023-10-24 Biontech Us Inc. Neoantigens and uses thereof
US12246067B2 (en) 2018-06-19 2025-03-11 Biontech Us Inc. Neoantigens and uses thereof
US11183272B2 (en) 2018-12-21 2021-11-23 Biontech Us Inc. Method and systems for prediction of HLA class II-specific epitopes and characterization of CD4+ T cells

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